Designation: D 3731 – 87 (Reapproved 2004) Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters1 This standard is issued under the fixed designation D 3731; the number immediately following the designation indicates the year of original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A superscript epsilon (e) indicates an editorial change since the last revision or reapproval. 1. Scope 4. Significance and Use 1.1 These practices include the extraction and the measure- 4.1 Data on the chlorophyll content of the algae have the ment of chlorophyll a, b, and c, and pheophytin a in freshwater following applications: and marine plankton and periphyton. Three practices are 4.1.1 To provide estimates of algal biomass and productiv- provided as follows: ity. 1.1.1 Spectrophotometric, trichromatic practice for measur- 4.1.2 To provide general information on the taxonomic ing chlorophyll a, b, and c. composition (major groups) of the algae, based on the relative 1.1.2 Spectrophotometric, monochromatic practice for mea- amounts of chlorophyll a, b, and c, and the physiological suring chlorophyll a corrected for pheophytin a; and for condition of algal communities, which is related to the relative measuring pheophytin a. abundance of pheopigments. 1.1.3 Fluorometric practice for measuring chlorophyll a 4.1.3 To determine long-term trends in water quality. corrected for pheophytin a; and for measuring pheophytin a. 4.1.4 To determine the trophic status of surface waters. 1.2 The values stated in SI units are to be regarded as the 4.1.5 To detect adverse effects of pollutants on plankton and standard. The values given in parentheses are provided for periphyton in receiving waters. information purposes only. 4.1.6 To determine maximum growth rates and yields in 1.3 This standard does not purport to address all of the algal growth potential tests. safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appro- 5. Interferences and Special Considerations priate safety and health practices and determine the applica- 5.1 Pigment Extraction—The chlorophylls are only poorly bility of regulatory limitations prior to use. Specific precau- extracted, if at all, from some forms of algae, such as the tionary statements are given in Section 7. coccoid green algae, unless the cells are disrupted, whereas other algae, such as the diatoms, give up their pigments very 2. Terminology readily when merely steeped in acetone. Since natural commu- 2.1 Definitions: nities of algae usually consist of a wide variety of taxa that 2.1.1 plankton—nonmotile or weakly swimming organisms, differ in their resistance to extraction, it is necessary to disrupt usually microscopic, that drift or are carried along by currents the cells routinely to ensure maximum recovery of the chloro- in surface waters, commonly consisting of bacteria, algae, phylls. Failure to do so may result in a systematic underesti- protozoa, rotifers, and microcrustacea. mation of 10 % or more in the chlorophyll content of the 2.1.2 periphyton—microorganisms growing on submerged samples. (1, 2, 3)2 objects, commonly consisting of bacteria, algae, protozoa, and 5.2 Grinders—The cells of many common coccoid green rotifers. algae resist disruption by most methods, but usually yield their pigments after maceration with a tissue grinder. The routine 3. Summary of Practices use of grinders, therefore, is recommended. Glass-to-glass 3.1 The chlorophyll and related compounds are extracted grinders are more rigorous in disrupting cells in plankton from the algae with 90 % aqueous acetone. The concentration concentrated by centrifugation, and in periphyton scrapings, of the pigments is determined by measuring the light absorp- than are TFE-fluorocarbon-to-glass grinders, and their use for tion or fluorescence of the extract. this purpose is preferred. However, TFE fluorocarbon-to-glass grinders perform well with glass-fiber filters. Other cell dis- 1 ruption methods, such as sonication, may be used if, for each These practices are under the jurisdiction of ASTM Committee E47 on Biological Effects and Environmental Fate and are the direct responsibility of Subcommittee E47.01 on Aquatic Assessment and Toxicology. 2 Current edition approved April 1, 2004. Published April 2004. Originally The boldface numbers in parentheses refer to the list of references at the end of approved in 1979. Last previous edition approved in 1998 as D 3731 - 79 (1998). this standard. Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States. 1 D 3731 – 87 (2004) type of sample, it is demonstrated that the chlorophyll recovery 6. Apparatus is comparable to that obtained with tissue grinders (4). 6.1 Filters, Glass-fiber filters, providing quantitative reten- 5.3 Filters—Glass-fiber filters usually provide a higher tion of particles equal to or greater than 0.45 µm in diameter. recovery of chlorophyll than is obtained with membrane filters 6.2 Filtering Apparatus suitable for use with glass-fiber when extraction-resistant algae are present in the samples, and filters. should be employed routinely (4). 6.3 Tissue Homogenizer—Tissue grinder consisting of a 5.4 Chlorophyll-Related Pigments—Naturally occurring, motor-driven pestle and enclosing glass tube (glass to glass or structurally related chlorophyll precursors and degradation TFE-fluorocarbon-to-glass grinder).4 products, such as the chlorophyllides, pheophytins, and 6.4 Spectrophotometer suitable for use over the range from pheophorbides, commonly occur in pigment extracts and may 600 to 750 nm, with a resolution of 2 nm or better, and absorb light in the same region of the spectrum as the equipped with sample cells having a light path of 1, 5, and 10 chlorophylls. These compounds may interfere with the analysis cm, with a capacity of 10 mL or less. by indicating falsely high chlorophyll concentrations. 6.5 Fluorometer (Optional): 5.4.1 This practice includes a correction for pheophytin a 6.5.1 Spectrophotofluorometer that provides an excitation only. Pheophytin a is similar in structure to chlorophyll a, but wavelength of 430 nm and detection of emission over the range lacks the magnesium atom (Mg) in the porphyrin ring. The from 600 to 700 nm, or: magnesium can be removed from chlorophyll in the laboratory 6.5.2 Filter Fluorometer equipped with a blue light source by acidifying the extract. When a solution of pure chlorophyll and blue excitation filter5 and a sharp cut off filter3 a is converted to pheophytin a by acidification, the absorption 6.6 Centrifuge that can provide a centrifugal force of 1000 peak is reduced to approximately 60 % of its original value and g; head with swing-out buckets preferred. shifts from 664 to 665 nm, resulting in a before:after acidifi- 6.7 Centrifuge Tubes, screw-cap or stoppered, conical, cation absorption peak ratio (OD664/OD665) of 1.70. This graduated, 15-mL. Avoid cap liners soluble in acetone and phenomenon is utilized in correcting the apparent concentra- neoprene rubber stoppers. tion of chlorophyll a for the presence of pheophytin a. Unwanted degradation of chlorophyll to pheophytin in the 7. Reagents and Materials phytoplankton on filters, or in periphyton samples, or in the 7.1 Aqueous Acetone, 90 %—Add 1 volume of distilled acetone extract, by the occurrence of acidic conditions can be water to 9 volumes of reagent grade acetone. Add 5 drops of 1 prevented by the addition of a magnesium carbonate suspen- N sodium bicarbonate solution per litre. (Caution—the vol- sion to the plankton sample before filtering or to the periphyton ume:volume relationship between the acetone and water must samples before grinding, and by adding a small amount of a be strictly followed to prevent shifts in the absorption peaks.) sodium bicarbonate solution to the aqueous acetone when it is 7.2 Hydrochloric Acid (1 N)—Add one volume of concen- prepared. Addition of magnesium carbonate may also aid in trated hydrochloric acid (HCl, sp gr 1.19) to eleven volumes of clarifying the samples following steeping (5). distilled water. 5.5 Turbidity—The optical density of the extract is mea- 7.3 Magnesium Carbonate Suspension—Add 1 g of finely sured at 750 nm to correct for turbidity. powdered magnesium carbonate to 100 mL of distilled water in 5.6 Spectrophotometer Resolution—The absorption peak of a stoppered Erlenmeyer flask. Shake immediately before use. acetone solutions of chlorophyll extracts is relatively narrow, 7.4 Sodium Bicarbonate Solution (1 N)—Prepare by dis- and a spectrophotometer with a resolution of 2 nm or better is solving 8.4 g of sodium bicarbonate in 100 mL of distilled required to obtain accurate results. If instruments of lower water. resolution are employed, the concentration of chlorophyll a may be significantly underestimated depending on the band 8. Sampling width. At a spectral band width of 20 nm, the error in the 8.1 Plankton: estimate of the chlorophyll a concentration may be as large as 8.1.1 Collection—Collect samples with a water bottle, dia- 40 %. phragm pump, or other suitable device. To protect the chloro- 5.7 Fluorometer Filters—In the fluorometric practice, inter- phyll from degradation prior to extraction and analysis, imme- ferences from light emitted by chlorophyll b and chlorophyll c diately add 1 mL of magnesium carbonate suspension per L of are greatly reduced by the use of a sharp cut-off red filter3 that sample, and protect from the direct sunlight. blocks all light with a wavelength of less than 650 nm (6). 8.1.2 Concentration—Immediately concentrate the plank- 5.8 Light Sensitivity of Extracts—Chlorophyll solutions de- ton by filtering or by centrifuging for 20 min at 1000 g. To grade rapidly in strong light. Work with these solutions, avoid cell damage and loss of contents during filtration, do not therefore, should be carried out in subdued light, and all exceed a vacuum of 1⁄2 atm (50 kPa). After centrifuging, check vessels, tubes, and so forth, containing the pigment extracts the samples for buoyant cells that may resist sedimentation. should be covered with aluminum foil or other opaque sub- stance. 4 Kontes type C, glass-to-glass grinder or its equivalent, has been found suitable for this purpose. Available from Kontes Manufacturing Co., Spruce St., Vineland, 3 Corning CS-2-64 filter or its equivalent, has been found suitable for this NJ 08360. 5 purpose. Available from Corning Glass Works, 388 Beartown Rd., Painted Post, NY Corning CS-5-60 filter has been found satisfactory. Equivalent filters may be 14870. used. 2 D 3731 – 87 (2004) 8.1.3 Holding Time—Samples that cannot be concentrated wavelengths, rather than the relative OD as is commonly the immediately after collection may be held at 0 to 4°C in the dark case in colorimetric analyses. For quantitative chlorophyll for 24 h before the plankton are concentrated. The centrifugate determinations, it is essential, therefore, to check the instru- or residue on the filter may be stored in the dark at −20°C for ment or chart OD readings, or both, at several wavelengths in 30 days before extracting the pigment. the range from 600 to 750 nm across the full span of the 8.2 Periphyton: absorbance (OD = 0.0–1.0), using a set of filters6 of known 8.2.1 Collection—Take samples from natural or artificial optical density. substrates and immediately ice, freeze, or place in cold (iced) 10.2.2 Transfer the extract to a sample cell and measure the aqueous acetone in the dark. optical density at 750, 664, 647, and 630 nm. If possible, 8.2.2 Holding Time—Iced samples may be held 24 h before choose a cell-path length or dilution to provide an OD664 they are further processed. Frozen samples may be held at −20°C for 30 days. greater than 0.2 and less than 1.0. 10.2.3 Subtract the OD750 from each of the other ODs. 9. Pigment Extraction Then divide by the cell-path length in centimetres. 9.1 Algal cells in plankton concentrates (from centrifuga- 10.2.4 Calculate the concentration of chlorophyll a, b, and c tion or filtration) and periphyton scrapings are disrupted by in the extract by inserting the 1-cm OD664, OD647, and grinding in a tissue homogenizer for 3 min at approximately OD630 into the following Jeffrey and Humphrey equations (6): 500 r/min in 4 to 5 mL of 90 % aqueous acetone. Use a Chl a, mg/L 5 11.85~OD664! 2 1.54~OD647! 2 0.08~OD630! glass-to-glass tissue grinder for macerating plankton concen- (1) trate obtained by centrifugation and for macerating periphyton scrapings. A TFE-fluorocarbon-to-glass or glass-to-glass Chl b, mg/L 5 21.03~OD647! 2 5.43~OD664! 2 2.66~OD630! (2) grinder may be used to macerate plankton concentrated on glass-fiber filters. Chl c, mg/L 5 24.52~OD630! 2 1.67~OD664! 2 7.60~OD647! 9.2 Wash the homogenate into a vial or into a 15-mL (3) centrifuge tube, rinse the pestle and grinding tube with a small 10.2.5 Express the concentration of pigments in a plankton amount of aqueous acetone, bring the volume of the extract to sample as milligrams per cubic metre (mg/m3) and calculate as 10 mL by adding 90 % aqueous acetone, cap or stopper the follows: tube, and mix and place the material in the dark at 4°C to steep. Ca 3 E 9.3 Steep not less than 15 min or more than 24 h. Mix the Chl a, mg/m 3 5 (4) G homogenate by inverting the tube several times, and clarify the extract by centrifuging 20 min at 1000 g, or by filtering. If the where: clarified extract is not analyzed immediately, store in the dark Ca = concentration of chlorophyll a in the extract, mg/L, at −20°C in a tightly stoppered tube. E = extract volumes, L, and 9.4 After clarification, decant the extract directly into a G = grab sample volume, m3. cuvette or a screw-cap or stoppered tube. If the analysis can not 10.2.6 Express the concentration of pigments in a periphy- be carried out immediately, the extract can be stored for 1 year ton sample as milligrams per square metre (mg/m2) and without appreciable chlorophyll degradation if held in the dark calculate as follows: at −20°C. Ca 3 E Chl a, mg/m 2 5 A (5) 10. Extract Analysis 10.1 The two practices of extract analysis commonly em- where: ployed are visible spectrophotometry and fluorometry (6, 7). Ca = concentration of chlorophyll a in the extract, mg/L, Each practice has its advantages and disadvantages. The and trichromatic, spectrophotometric practice, has the advantage of A = substrate area sampled, m2. providing a simple procedure for the simultaneous estimation 10.3 Spectrophotometric, Monochromatic Practice: of chlorophyll a, b, and c, which, at the current state of 10.3.1 Measure the optical density of the chlorophyll extract technology, cannot be obtained as easily by the fluorometric practice, but does not correct for chlorophyll degradation at 750 and 664 nm. products. The monochromatic, spectrophotometric practice 10.3.2 Acidify the extract by adding 2 drops of 1 N HCl corrects the chlorophyll a for pheophytin, a, but does not (amount added to a 1-cm cell; if a larger cell is used, add a measure chlorophyll b and c. The fluorometric practice, is one proportionately larger volume of acid), stir well, and measure to three orders of magnitude more sensitive than the spectro- the OD at 750 and 665 nm not sooner than 1 min or later than photometric practices, when using the instrumentation com- 2 min after acidification. monly employed for chlorophyll analyses, but the practices for simultaneously measuring chlorophyll b and c are much more complex. 6 Filters, Standard Reference Material 930, Glass Filters for Spectrophotometry, 10.2 Spectrophotometric, Trichromatic Practice: available from the National Bureau of Standards, Office of Product Standards, 10.2.1 The chlorophyll concentration is a function of the Administration Building A603, Gaithersburg, MD 20899, or equivalent have been absolute optical density (OD) of the extract at the specified found suitable for this purpose. 3 D 3731 – 87 (2004) 10.3.3 Calculate the concentration of chlorophyll a (Ca) where: corrected for the presence of pheophytin a, and the concentra- Ca = concentration of chlorophyll a, µg/L, and tion of pheopigments expressed as pheophytin a (Pa) in the R = fluorometer reading. extract by inserting the 1-cm ODs in the following equations 10.4.3 To correct for the presence of pheopigments, ex- (5): pressed as pheophytin a, determine a before:after acidification Ca, mg/L 5 26.7~OD664b 2 OD665a! (6) fluorescence ratio, r, as described in 10.3.2 using an extract that is free of pheophytin a, where the before:after acidification Pa, mg/L 5 26.7[1.7~OD665a! 2 OD664b# (7) ratio is 1.70, based on the OD664b/OD665a, as determined with a spectrophotometer. Use the fluorometric before:after where: acidification ratio (r) and the calculation factor, F (s), in the OD664b = OD664 − OD750 measured before acidifica- following equations: tion, and r OD665a = OD665 − OD750 measured after acidification. Ca, µg/L 5 F~s! r 2 1 ~Rb 2 Ra! (9) 10.3.4 Calculate the concentration of these pigments in plankton and periphyton samples as described in 10.2.5 and 10.2.6, respectively. r Pa, µg/L 5 F~s! r 2 1 ~rRa 2 Rb! (10) 10.4 Fluorometric Practice: 10.4.1 The fluorometric practice is 10 to 1000 times more sensitive than the spectrophotometric practices and requires where: Ca and Pa = concentration of chlorophyll a and pheophy- proportionately smaller amounts of sample. The method has tin a, respectively, in the extract, important disadvantages, however, which include the inability Rb = fluorometer reading before acidification; and to easily determine chlorophyll b and c concentrations, and the Ra = fluorometer reading after acidification. need to calibrate the instrument with “reference” chlorophyll solutions containing a known concentration of chlorophyll a 11. Precision and Bias determined by previous spectrophotometric analysis (7). 10.4.2 To calibrate the instrument, carefully dilute the 11.1 It is not practicable to specify the precision of the “reference” extract to provide solutions that give midscale procedures in Practices D 3731 for measuring chlorophyll readings in each sensitivity range of the fluorometer. Use the content of algae since there are no interlaboratory data sets readings to determine a calibration factor, F (s), for each available at this time. sensitivity level (s) as follows: Ca 12. Keywords F~s! 5 R (8) 12.1 algae surface water; chlorophyll; pheophytin REFERENCES (1) Golterman, H. L., “Methods for Chemical Analysis of Fresh Waters,” Limnology and Oceanography, Vol 16, No. 6, 1971, pp. 990–992. International Biological Program Handbook No. 8. Blackwell Scien- (5) Lorenzen, C. J., “Determination of Chlorophyll and Phaeopigments: tific Publication, London, 1969, p. 172. Spectrophotometric Equations,” Journal of Limnology and Oceanog- (2) Strickland, J. D. H., and Parsons, T. R., “A Practical Handbook of raphy, Vol 12, pp. 343–346. Seawater Analysis,” Bulletin Fisheries of the Research Board of (6) Jeffrey, S. W., and Humphrey G. F., “New Spectrophotometric Canada, No. 167, Ottawa, 1968, p. 311. Equations for Determining Chlorophylls a, b, c1 and c2 in Higher (3) Vollenweider, R. A., ed., “A Manual on Methods for Measuring Plants, Algae, and Natural Phytoplankton,” Biochemistry and Physi- Primary Production in Aquatic Environments,” International Biologi- ology of Plants, Vol 167, ZEB Gustav Fischer Verlag, East Germany, cal Program Handbook No. 12, Second Edition, Blackwell Scientific 1975, pp. 191–194. Publication, London, 1974, p. 225. (7) Holm-Hansen, O., Lorenzen, C. J., Holmes, L. W., and Strickland J. D. (4) Long, E. V., and Cooke, G. D., “A Quantitative Comparison of H., “Fluorometric Determination of Chlorophyll,” Journal of Cons. Pigment Extraction by Membrane and Glass-Fiber Filters,” Journal of Int. Explor. Mer. Vol 30, No. 1, pp. 3–15. ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned in this standard. 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